Regional thermal variation in a coral reef fish

This study investigated the thermal performance of several traits from damselfish populations in two different regions on the Great Barrier Reef (low- and high-latitude). Oxygen consumption rates and aerobic enzyme activity at elevated temperatures were found to differ between the regions, while immunocompetence, haematocrit, and anaerobic enzyme activity were similar.


Introduction
The response of species to climate change is determined by the collective response of populations (Bennett et al., 2019;McKenzie et al., 2021).How populations respond to environmental change can vary over geographic and environmental gradients, due to variation in traits that have evolved via genetic adaptation and phenotypic plasticity (Sorte et al., 2011;Des Roches et al., 2018;Bennett et al., 2019;Plumb et al., 2020).Temperature conditions, particularly amongst ectotherms, are hypothesized to produce macrobiogeographical patterns that reflect thermal constraints on organisms' biochemistry and physiology (Somero, 2010;Pereira et al., 2017).Co-gradient variation across thermal clines, whereby genetic and environmental influences on phenotype are aligned (e.g.populations exposed to higher temperatures have higher optimal performance temperatures), has been demonstrated in a variety of taxa (plants: Aitken and Bemmels, 2016;Mahony et al., 2020, insects: Hoffmann et al., 2003;Barton et al., 2014, crustaceans: Kuo andSanford, 2009;Sorte et al., 2011;Yampolsky et al., 2014 and fish: see review by Conover et al., 2009).However, optimal performance temperatures often do not follow the trajectory of environmental gradients (Conover et al., 2009).Countergradient variation occurs when phenotypes possess greater plastic responses than phenotypic divergence between populations, or phenotypes diverge in the opposite direction of environmental gradients (Conover and Schultz, 1995;Schmid and Guillaume, 2017;Stamp and Hadfield, 2020).Countergradient variation has been recorded in several taxa (lizards: Angilletta et al., 2004;Hodgson and Schwanz, 2019, turtles: Snover et al., 2015and fish: Gardiner et al., 2010); however, the extent to which phenotypic plasticity and genetic differentiation contribute to countergradient variation differs (Stamp and Hadfield, 2020).
Population responses to warming temperatures will depend on their occupied thermal niche.Low-latitude environments characterized by stable temperatures near physiological maximums favour specialized (narrow) thermal niche breadths that primarily evolved through genetic adaptation (i.e.selection for particular phenotypes) rather than plasticity-Climate Variability Hypothesis (Janzen, 1967;Stevens, 1989; but see Overgaard et al., 2011;Chiono and Paul, 2023).Narrow thermal niche breadths, limited plasticity, and evidence of hard ceilings for upper thermal tolerance (Gunderson and Stillman, 2015;Sandblom et al., 2016;Morgan et al., 2020) suggest that low-latitude populations are more vulnerable to shifting temperatures than high-latitude populations (Stillman, 2003;Deutsch et al., 2008;Tewksbury et al., 2008;Somero, 2010;Sunday et al., 2011).High-latitude populations that experience variable environmental conditions are predicted to retain greater benefits from phenotypic plasticity than populations living in thermally stable environments (Janzen, 1967;Stevens, 1989); nonetheless, empirical evidence remains scarce (but see, Molina-Montenegro and Naya, 2012;Naya et al., 2012;Donelson et al., 2019).Wider thermal niche breadths have been reported in high-latitude populations (Sunday et al., 2011;Shah et al., 2017;Stuart-Smith et al., 2017;McKenzie et al., 2021), however, heat-tolerant phenotypes present in low-latitude populations may be unattainable within highlatitude populations (Kelly et al., 2012).Individual populations may therefore possess thermal niches that are narrower than the species as a whole (Kelly and Griffiths, 2021).
Intraspecific variation in thermal performance within marine systems has not received the same attention as terrestrial systems, despite marine organisms having greater confinement to thermal tolerance limits (Sanford and Kelly, 2011;Sunday et al., 2011;Pinsky et al., 2019;Lenoir et al., 2020).Within terrestrial systems local adaptation is already being incorporated into conservation considerations to prepare organisms for projected climate change scenarios (Aitken and Whitlock, 2013;Aitken and Bemmels, 2016;Liepe et al., 2016;Bazzicalupo et al., 2023).Marine systems hereinto have been demographically viewed as well-connected networks where locally adapted traits are expected to be overwhelmed by gene flow.However, a growing body of evidence suggests that oceanographic features, life history traits and larval dispersal ability can act as challenges to gene flow and promote local adaptation (Jones et al., 1999;Swearer et al., 2002;Sanford and Kelly, 2011).Evidence of local adaptation and how it impacts the ability to predict species responses to climate change has been demonstrated amongst marine crustaceans (Stillman, 2002;Kuo and Sanford, 2009;Sorte et al., 2011;Kelly et al., 2012;Pereira et al., 2017;Sasaki and Dam, 2019) and corals (van Oppen et al., 2014); yet few studies broach the topic amongst marine fish.
Thermal intraspecific variation in marine fishes may differ depending on larval dispersal ability and local environmental conditions; therefore, broadscale geographical patterns such as the climate variability hypothesis and co-/countergradient variation are unlikely to be universally applicable (Calosi et al., 2008;Pereira et al., 2017;Sasaki and Dam, 2019).A case study comparing low-and high-latitude populations of coral trout (Plectropomus leopardus), a species with a pelagic larval stage and high level of population connectivity (via spatial and temporal variation in larval recruitment; Van Herwerden et al., 2009;Taboun et al., 2021), found no significant differences in physiological metrics between populations (Pratchett et al., 2013).However, patterns of countergradient variation and genetic distinctness have been identified amongst marine fish species with high-(Gadus morhua; Marcil et al., 2006) and low-dispersal (Acanthochromis polyacanthus; Gardiner et al., 2010;Donelson and Munday, 2012) ability between populations.The lack of uniformity in broadscale geographic patterns amongst marine fish necessitates the examination of population-based responses.
Genetic differentiation and intraspecific variation in thermal physiology of the coral reef damselfish A. polyacanthus is evident; however, existing physiological studies provide a coarse understanding of differences between populations.The inability for A. polyacanthus to disperse through water channels greater than ∼10 m has created a genetically isolated and heterogenous mixture of populations (reefs) that are strongly influenced by founder effects (Doherty et al., 1994;Planes et al., 2001;Miller-Sims et al., 2008).Between different regions, and even neighbouring reefs, populations display divergence at the nuclear level and lack the ability to exchange genes in the absence of connecting shallow channels (Doherty et al., 1994;Planes et al., 2001).Hereinto, physiological data from high-latitude populations emanates from a single lagoonal population that experiences high levels of diurnal and seasonal temperature fluctuations compared to most reef systems (i.e.Heron Island; Gardiner et al., 2010;Donelson and Munday, 2012).When comparing wild-sourced A. polyacanthus from Heron Island and low-latitude populations (i.e.Lizard Island, Palm Island) oxygen consumption rates at elevated temperatures were found to be similar between regions (Donelson and Munday, 2012) or more efficient amongst fish from Heron Island (Gardiner et al., 2010)-countergradient variation.Results on equatorial A. polyacanthus populations (Papua New Guinea), following comparative methods to Gardiner et al. (2010), found complementary evidence that optimal temperatures for oxygen consumption were similar between equatorial, low-latitude and high-latitude populations (Rummer et al., 2014) low-latitude regions may therefore be more susceptible to warming ocean temperatures than high-latitude populations as they are already living closer to their thermal limits (Rummer et al., 2014).However, these results come from the sampling of just four populations across A. polyacanthus's range, and it remains unclear how representative, and applicable, these results are to neighbouring populations or throughout the species range.
The objective of this study was to increase the resolution of A. polyacanthus's thermal landscape and allude to a finer understanding of existing intraspecific variation within marine environments.Thermal performance curves of key physiological traits in A. polyacanthus were compared using three different reefs amongst two regions of the Great Barrier Reef (GBR), low latitude (∼Cairns) and high latitude (∼Mackay), that experience different thermal profiles.Based on previous studies, we hypothesized that high-latitude populations that experience greater thermal variation will possess greater physiological performance at elevated temperatures than low-latitude populations (i.e.countergradient variation).At elevated temperatures, fish from higher latitudes were expected to outperform fish from low latitudes when measuring physiologically informative metrics including aerobic physiology (i.e.oxygen consumption), immunocompetence, haematocrit, and enzyme activity.Tested physiological metrics are associated with energetic demanding processes that have previously been identified as being sensitive to environmental temperatures (Supplementary Table S1).Additionally, co-gradient variation remains a valid alternative hypothesis considering previously demonstrated genetic differentiation, lack of variability in studied populations and the unique nature of the previously tested Heron Island population.

Materials and Methods
This research was completed under James Cook University ethics approval A2764.

Study species
The tropical damselfish, A. polyacanthus (Bleeker, 1855), ranges from the Philippines to the southern end of the GBR (∼15 • N to ∼23 • S; Allen, 1991).Acanthochromis polyacanthus populations are hypothesized to have propagated throughout the Indo-Pacific proceeding the Pleistocene (2.6 Ma-11.7 ka) as rising sea levels reestablished dispersal corridors between reefs (Van Herwerden and Doherty, 2006;Ludt and Rocha, 2015).However, such dispersal corridors dissipated as sea levels reached present-day conditions (Miller-Sims et al., 2008).Acanthochromis polyacanthus lacks a pelagic larval development period, instead performing parental care during embryonic and early life development, in socially monogamous pairs, until fry are large enough to disperse into the surrounding habitat (Robertson, 1973).Dispersal is limited to adjacent reefs separated by depths less than ∼10 m, creating conditions where reefs that are not connected by shallow channels are genetically isolated and differentiated from one another (electrophoresis, Doherty et al., 1994;microsatellite markers, Miller-Sims et al., 2008).Acanthochromis polyacanthus are ideal for examining local adaptation in marine environments as they possess a broad geographic distribution, across thermally variable environments, where gene flow is limited.

Sampling
Adult A. polyacanthus were collected via professional collectors from June to December 2021 from six different reefs and two different regions (low-and high-latitude) that were absent of shallow channels, which would have allowed dispersal between populations.Three reefs from low-latitude locations were sampled, including Tongue Reef (−16.341  1).Low-and high-latitude collection sites are separated by ∼400 km (spanning ∼5 • in latitude).In total, 55 fish were sampled over the duration of the experiment (Supplementary Table S2).Of the initial 55 fish, 38 completed all experimental assays including: resting oxygen consumption, maximum oxygen consumption, absolute aerobic scope, immunocompetence, haematocrit, and enzyme activity.Seventeen fish experienced mortality events at various stages of the experiment.
Adult fish were held in separate 52-l opaque aquariums (56 × 35 × 30 cm) inside an environmentally controlled aquarium room at the Marine and Aquaculture Research Facility at James Cook University (Townsville, Australia).Each aquarium contained a shelter (half a terra-cotta pot), constant aeration and water flow (2 l min −1 ) at set experimental conditions (see below).Fish were transferred to the experiment room that was used for trials on 25 May 2022.Respirometry and immunity trials occurred from 6 June to 17 August 2022.Tissue (enzymes) and blood (haematocrit) samples were collected on 1 September 2022, 2 weeks after respirometry and immunity trails concluded.A random number generator was used to determine the order and chamber fish were tested in.

Thermal conditions
To understand local thermal conditions for reefs within lowlatitude and high-latitude regions, temperature data was collected via the Australian Institute of Marine Science (AIMS) temperature logger data series.Temperature data was collected from loggers at depths of 7-15 m, for a subset of reefs (Supplementary Table S3) from each region (AIMS 2020; Supplementary Fig. S1).Experimental temperatures used for repeated oxygen consumption and immunocompetence thermal performance curves included the approximate daily mean summer temperature for both high-latitude (∼27 • C) and lowlatitude (∼28.  ) low-and high-latitudinal regions that fish were collected from across the Great Barrier Reef.Black points represent sites where fish were sampled in previous research, including Lizard Island (L), Palm Island (P) and Heron Island (H).Insert B) provides a zoomed-in perspective of the low-latitude region that was made up of fish from three different reefs including Sudbury Reef, Tongue Reef and Vlasoff Reef.Insert C) provides a zoomed-in perspective of the high-latitude region that is made up of two inshore islands, Cockermouth and Keswick Island, and one offshore reef, Chauvel Reef (southern).mid-century, IPCC climate change scenarios project global surface temperature changes of +1.5 • C by mid-century; two of these scenarios, SSP3-7.0 and SSP5-8.5, project > +3.0 • C by 2100 (IPCC, 2021).For low-latitude populations 30 and 31.5 • C represent projected mid-and end-of-century conditions; additionally, 31.5 • C represents approximate presentday maximum summer temperatures.For high-latitude populations 28.5 and 30 • C represent projected mid-and end-ofcentury conditions, with 30 • C also representing approximate present-day maximum summer temperatures and 31.5 • C representing a temperature that is presently not experienced.Testing began at the coolest temperature of 27 • C. Fish were given at least 48 h to rest between aerobic physiology and immunocompetence trails.Once oxygen consumption and immunocompetence testing was completed for a given temperature, fish were warmed to the next temperature (+1.5 • C), at a rate of +0.5 • C day −1 for three consecutive days.Fish were then provided with an additional 5 days to adjust to the new temperature before the next sampling period began.Sampling periods took place over 4-5 days.This process was repeated for all testing temperatures.

Aerobic physiology
Resting and maximum oxygen consumption were determined via intermittent flow respirometry.Chambers were 1.5 l in volume and custom built from PVC pipe and acrylic (Supplementary Fig. S2).The experimental setup consisted of two temperature-controlled aquaria (260 l), with continuous water exchange and aeration, each containing four submerged respirometry chambers placed in parallel.Chambers were opaque except for the lid, so that fish could not view each other, but light could still enter the chamber.Each respirometry chamber unit contained an independent brushless DC recirculation pump (flow rate 240 l h −1 ), vinyl tubing (composing ∼1% of the total water volume) and an inline oxygen sensor probe (multichannel FireSting-O2, PyroScience GmbH, Aachen, Germany).Oxygen sensor probes were calibrated to 0% air, using sodium sulphite (Na 2 SO 3 )-saturated seawater, at the beginning of the experiment and when spot material was replaced.One hundred percent air calibrations were conducted at the beginning of each trial.During flush periods a pump (AQUAPRO, AP750LV; 750 l h −1 ) was used to flush each set of four chambers simultaneously.Heaters (2 kW) and temperature sensors (Semitec 103AT-11 IP67) were used to ensure that experimental temperatures remained within ±0.3 • C of experimental temperature set points.Minimal background respiration was achieved through UV filtration, particle filtration (100-μm bag filters) and daily cleaning of equipment (bleach diluted to 200 ppm with fresh water).Fish were deprived of food for 18-24 h before respiration trials began (Chabot et al., 2016).Trials were conducted in a fully lit room to eliminate metabolic costs associated with photoperiod (Chabot et al., 2016).
Maximum oxygen consumption (MO 2max ) was used as a proxy for maximum metabolic rate (Norin and Clark, 2016).To achieve maximum oxygen consumption fish were placed in a swim tunnel for 10 min.During the initial 5-min interval, the speed of water flow through the swim tunnel was slowly increased until fish displayed a change in gait swimming behaviour, defined as a transitioning behaviour from predominately pectoral swimming to body/tail undulations (Supplementary Video 1).The speed of the swim tunnel that produced this intermediary transitional swimming behaviour was maintained for the second 5-min interval.Immediately after the 10-min swimming period, fish were collected by hand and transferred to a randomly selected respiration chamber.Pilot studies (unpublished data, Schmidt) determined that highest MO 2max levels were achieved with the immediate transfer of fish from the swim tunnel to respiration chambers, rather than including an intermediary air exposure period.Therefore, no extended air exposure time was included prior to fish being transferred into respiration chambers.The time between fish being placed in respiration chambers and data being recorded (i.e.start of the wait period) was <10 s.The MO 2max was measured over 30-s intervals via rolling regressions within the 'auto_rate' function included in the R package 'respR' (v2.0.1;Harianto et al., 2019).The steepest slope (highest oxygen consumption rate) with an r 2 threshold of 0.95 was used to determine MO 2max .The MO 2max was measured prior to resting metabolic rate (MO 2rest ).Fish were held in respirometry chambers for 3.5-6 h (μ = 4.67 h) to measure MO 2rest .Measurement times for MO 2rest were based off previous experiments conducted on A. polyacanthus (Nilsson et al., 2009;Donelson and Munday, 2012;Bernal et al., 2018).Additionally, amongst small warm water fish, Rummer et al. (2014) found no deviation or continued decrease in oxygen consumption rates after 90 min of fish being placed in respiration chambers.Oxygen consumption was measured continuously over cycles consisting of a 15-s wait, 225-s measurement and 180-s flush period.Air percentage never dropped below <80% air saturation.Oxygen consumption rates were measured over a 220-s interval with an r 2 threshold of 0.95.The MO 2rest was measured by taking the mean of the lowest three oxygen consumption slopes.Background respiration was measured at the start of each trial by measuring oxygen consumption within empty chambers for at least three consecutive cycles.Background respiration levels typically accounted for <2% of measured oxygen usage rates and were therefore ignored.The mass of fish was measured at the end of all respiratory trials, after fish had been euthanized and patted dry with paper towels to avoid the inclusion of excess moisture.The mean fish-to-chamber volume ratio was 1:60 (Supplementary Fig. S3) but varied depending on the size of each fish.Oxygen consumption rates were converted from percent air saturation values to milligrammes per hour via the 'convert_rate' function within the R package respR (Harianto et al., 2019).Absolute aerobic scope (AAS) was calculated by subtracting MO 2rest from MO 2max.

Immune response
To test immunocompetence, subcutaneous phytohaemagglutinin injections were used to produce a (localized) cell-mediated response in vivo (e.g.inflammation, T-cell proliferation, infiltration of immune cells; Martin et al., 2006;LaMonica et al., 2021; pilot study on A. polyacanthus conducted by Donelson and Yasutake, 2024).Tissue swelling 24 h post-injection is mediated via complex immunological cascade.However, this tissue swelling is primarily driven via the congregation of leukocytes to the injection site (Martin et al., 2006).Fish were injected in the caudal peduncle subcutaneously with 0.03 ml of phytohaemagglutinin (PHA; L8754 Sigma-Aldrich, 45 ug 10 ul −1 ) dissolved in phosphate buffer saline (PBS), made to a ratio of 1 mg PHA to 1 ml PBS.The immunocompetence of fish was determined by measuring the width of the injection area with pressure-sensitive callipers (Mitutoyo ABS Digimatic; accuracy 0.1 mm) pre-injection and ∼18-24 h post-injection.The difference in localized swelling pre-and post-injection was used as a proxy for immunocompetence.

Fish tissue sampling
Whole blood and white muscle tissue samples were collected 10 days after all oxygen consumption and immunocompetence trails were completed at the final testing temperature (31.5 • C).Whole blood was collected from the caudal vein via heparin-coated 25-gauge surgical needles.Fish were then euthanized via cervical dislocation.White muscle tissue samples were dissected from tissue between the dorsal fin and lateral line.Once obtained, tissue samples were stored in liquid nitrogen and then transferred to a −80 • C freezer.60 s to separate red cells from blood plasma.The proportion of blood volume occupied by red blood cells (haematocrit) was recorded by using a ruler to first measure the space of the microcapillary tube that was occupied by the total blood volume (packed red blood cells and blood plasma), followed by measuring the space occupied by packed red blood cells.Haematocrit scores were calculated using the following formula: hematocrit = packed red blood cells total blood volume

Enzyme activity
White muscle tissue was used to examine the maximal enzyme activity of lactate dehydrogenase (LDH) and citrate synthase (CS).Testing temperatures of 20, 30, 40 and 50 • C were used to measure enzyme activity and the associated thermal performance curve.White muscle tissue was used for measuring enzyme activity analysis because its anaerobic capacity has been shown to correlate to whole-organism oxygen consumption, it contributes to the largest fraction of body mass and plays an important role in high-speed burst swimming (Sullivan and Somero, 1980).
The protocol used to measure enzyme activity method used was adapted from previous studies (Thibault et al., 1997;Seebacher et al., 2003;Lang et al., 2021).Samples were defrosted on ice.A sterile scalpel blade was used to extract a tissue sample (20-40 mg).Extracted tissue samples were homogenized via a microtube homogenizer (BeadBug 3, Benchmark Scientific, Model D1030-E) in a 1:10 dilution with a buffer consisting of 50 mmol l −1 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), 1 mmol l −1 ethylenediaminetetraacetic acid (EDTA), 0.01% Triton X-100 and 99.99% Milli-Q water, and adjusted to pH 7.4 with sodium hydroxide (NaOH).A subset of homogenized tissue was extracted for LDH and CS.Homogenized tissue samples used for the LDH assay were centrifuged (Eppendorf Centrifuge 5424, Hamburg, Germany) at 150 rpm for <3 s.Homogenized tissue samples used for the CS assay were not centrifuged to allow mitochondria to be retained.
Absorbance readings were measured with a spectrophotometer every 2 s, with 20 readings over 13 min (UV5, Mettler-Toledo, Columbus, OH).Testing temperatures were maintained with a Loop L100 circulation thermostat (Lauda, Lauda-Königshofen, Germany).All samples were measured in triplicate and included a positive and negative control.
The mean slope was used to determine enzyme activity.Background activity was subtracted from sample absorbance slopes when background activity exceeded 5% of sample absorption levels.Absolute enzyme activity levels were calculated in units per milligramme tissue (U mg −1 tissue) using the following formula where: A represents the absolute mean absorption of tested sample in triplicate, L represents the light path length (centimetre), represents the molar absorptivity/extinction coefficient (M −1 cm −1 ), c represents tissue sample concentration (mg/ml), and V represents volume.

Statistical analysis
Fish from different populations were grouped together based on whether they were collected from low-(Sudbury Reef, Tongue Reef and Vlassof Cay) or high-latitude (Cockermouth Island, Keswick Island and Chauvel Reef) reefs.From here onwards these groups will be referred to as low-or highlatitude regions.Generalized linear mixed effect models were used to test for differences in thermal performance curves associated with oxygen consumption, immunocompetence, and enzyme activity between low-and high-latitude populations (Supplementary Table S4).Model comparison and selection were performed using Akaike information criterion (AICc), Bayesian information criterion (BIC), and r-squared values (see software and packages section for more information).For thermal performance curves, model selection was used to determine if temperature should be modelled as a first, second or third order polynomial.Model selection was also used to compare alternative hypothesis-driven models (see 'Software' section in Methods for details).Statistical significance of independent variables and interactions was determined via likelihood-ratio chi-square values.Significant differences in post hoc analysis was determined by using estimated marginal means.Individual identification codes for each fish were used as a random factor due to repeated measures when modelling oxygen consumption, immunocompetence, and enzyme activity thermal performance curves.
All oxygen consumption models were run using a Gaussian distribution.To model oxygen consumption, including MO 2rest , MO 2max , and AAS, independent variables including latitude and temperature were modelled as fixed factors with an interaction term.Fish mass (centred) was positively related to oxygen consumption and therefore used as a covariate within oxygen consumption models.All oxygen consumption traits were modelled with temperature as a continuous second order polynomial.The model for MO 2rest included the additional covariate of testing runtime.Immunocompetence was modelled as a function of latitude, temperature (third order polynomial) and their interaction term.Additionally, a gamma distribution (with a log-link function) was used instead of a Gaussian distribution.Fish mass was not included as a co-variate within the immunocompetence model.
Haematocrit was modelled via a Gaussian distribution as a linear regression with latitude as the only independent variable.Haematocrit was only measured at a single temperature (31.5 • C).No random factor was included within the haematocrit model.
When modelling enzyme activity for LDH, CS, and the ratio of LDH:CS, a Gaussian distribution with an interaction between latitude and temperature, as well as sample tissue mass (centred) as a co-variate, was used.Additionally, citrate synthase was modelled with a log-link function.Temperature was modelled as a continuous second order polynomial for LDH and CS, and linearly for LDH:CS.

Haematocrit
Whilst there was a trend of slightly higher haematocrit level in high-latitude populations, no significant difference was observed in haematocrit levels between low-and highlatitude populations at 31.5 • C (χ 2 = 3.84, df = 1, P = 0.058; Supplementary Fig. S4).Packed red blood cells composed 22.4 and 25.9% of whole blood for low-and high-latitude populations, respectively.

Discussion
How populations respond to climate change will depend on traits that are adapted to localized environmental conditions.Localized environmental conditions can influence thermal preferences and limits within populations via plastic and evolutionary mechanisms, creating a complex adaptive landscape across species' distributions (Huey et al., 2012;Valladares et al., 2014).Identifying existing intraspecific variation is therefore essential to accurately predict populations' (and therefore species') responses to climate change.Our study found evidence of co-gradient variation (i.e.phenotypes align with the observed thermal gradient) within oxygen consumption, absolute aerobic scope, and CS (aerobic) enzyme activity, suggesting that these traits are adapted to localized environmental conditions.However, no intraspecific variation was found in several other traits including immunocompetence, haematocrit, and LDH enzyme activity.
Evidence of co-gradient variation was observed in maximum oxygen consumption and absolute aerobic scope.Fish from the low-latitude region displayed a higher capacity for MO 2max and AAS at 30 and 31.5 • C, compared to highlatitude conspecifics.Fish from the low-latitude region exhibited rising MO 2max and MO 2rest with warming; however, high-latitude populations displayed a plateaued MO 2max across the testing temperature range and consequently slightly reduced AAS, due to increasing MO 2rest .Increased absolute aerobic scope at higher temperatures suggests fish from the low-latitude region are currently better equipped to respond to warmer temperatures (within generation), compared to high-latitude conspecifics.According to the Oxygen and Capacity Limitation of Thermal Tolerance (OCLTT; Pörtner and Knust, 2007), AAS can serve as a proxy for the limits of oxygen-demanding processes (e.g.motor activity, reproductive output, growth) that can be performed simultaneously and is expected to be a primary mechanism that determines how fish will respond to climate change (Pörtner et al., 2017).However, the OCLTT hypothesis remains contested within the literature (Clark et al., 2013;Lefevre et al., 2021).To further understand how results from this study related to fitness and performance under projected future conditions, additional studies should aim to link interpopulation differences in MO 2max and AAS with reproductive metrics, as well as consider the potential to rapidly shift phenotypes via plasticity.
Despite differences in AAS between regions there was no significant difference in haematocrit.Stronger selection pressures on alternative traits, balancing selection, and/or physiochemical limitations may provide a mechanism for why no significant differences were identified between lowand high-latitude regions for these traits.For example, when examining thermal regulation behaviour in high-and low-elevation populations of the jacky lizard (Amphibolurus muricatus), Hodgson and Schwanz (2019) found significant differences in panting behaviour, but not basking intensity or duration.Immune response within several species, including Drosophilia melanogaster (Early and Clark, 2017) and three-spined stickleback (Gasterosteus aculeatus; Robertson et al., 2017), has been characterized by purifying selection and a lack of local adaptation.Considering the observed pattern in AAS, we might have expected latitudinal differences in haematocrit (a proxy for oxygen-carrying capacity of circulatory system; Gallaugher et al., 1995).In the case of the coral reef snapper (Lutjanus carponotatus), exposure to a marine heatwave of 29.5 and 30.5 • C (+1-2 • C) for 4 weeks resulted in an increase in haematocrit to allow maintenance of aerobic capacity (McMahon et al., in review).However, haematocrit was shown to be unresponsive in both the fusilier Caesion cuning and the cardinalfish Cheilodipterus quinquelineatus when exposed to elevated temperatures (+3.0 • C above ambient temperature) for 5 weeks (Johansen et al., 2021).
CS activity was significantly higher in the high-latitude populations compared to the low-latitude regions and may represent a potential adaptation to cooler environmental conditions.As temperatures shift away from thermal optima, towards cooler temperatures, the ability to produce ATP can be inhibited via decreased rates of chemical reactions, rates of diffusion and membrane fluidity (Lucassen et al., 2006;Dhillon and Schulte, 2011).To offset ATP deficiency in cooler environments conspecifics have increased mitochondrial volume density compared to warmer latitude conspecifics (Guderley, 2004).This co-gradient pattern has been observed across closely related species (Johnston et al., 1998, various Perciformes), as well as conspecifics from cold and warm environments (Lucassen et al., 2006; Gadus morhua).The pattern of overall higher CS (proxy for mitochondrial volume density) activity in high-latitude populations compared to low-latitude populations suggests that enzymatic maximum respiratory capacity is an unlikely candidate for directly causing whole-organism failure.Growing evidence suggests that whole-organism failure is not driven by a single mechanism, but rather can occur through multiple different mechanisms that can vary between species and contexts (Ern et al., 2023).However, common patterns have emerged suggesting major mechanisms such as ATP synthetic capacity, proton leakage, and the buildup of reactive oxygen species (i.e.indirect results of aerobic respiration) may be more relevant at upper thermal limits (Chung and Schulte, 2020;Ern et al., 2023), particularly within cardiac tissue that has a clear functional link with whole-organism performance (Farrell, 2009;Eliason et al., 2011;Ekström et al., 2017;Nyboer and Chapman, 2018;Pichaud et al., 2019).However, testing upper thermal tolerance mechanisms within cardiac muscle of coral reef fish remains challenging due to size of tissue available for sampling.
No significant differences were identified in the thermal performance curves of LDH or LDH:CS when comparing fish from low-and high-latitude regions.LDH and CS are proxy representations for anaerobic glycolysis and aerobic capacity that be can achieved via the citric acid cycle, respectively (Dahlhoff, 2004;Savoie et al., 2008;Ekström et al., 2017;Pichaud et al., 2019).The positive relationship between temperature and LDH:CS ratios indicate that as temperatures warm there is a greater reliance on anaerobic metabolism, a pattern that is expected amongst ectotherms, and which has previously been identified in several taxa including, crownof-thorns sea stars (Lang et al., 2021;Acanthaster spp.), dogfish sharks (Bouyoucos et al., 2023; Squalus suckleyi), Antarctic notothenioids (Mark et al., 2002;Jayasundara et al., 2013) and coral reef fish (Illing et al., 2020;Johansen et al., 2021).However, increased reliance on anaerobic metabolism can become unstable over long periods of time, due to the availability of finite fermentable substrates and cytotoxicity (Rosa et al., 2016).
Whilst there was no latitudinal difference in immunocompetence, there was a negative relationship with temperature.If the reduced swelling at elevated temperatures was due to a lack of available energy, A. polyacanthus may be immunocompromised prior to impacts on aerobic capacity, especially in the low-latitude region.A similar response has been observed in another coral reef fish from a similar latitude, the rabbitfish Siganus doliatus, where immunocompetence was negligible at 31.5 • C (LaMonica et al., 2021).Whilst immunological research in fish is emerging and scarce compared to other taxa, within bird species PHA swelling responses have been shown to be less costly than other activities (e.g.moulting, breeding; Martin et al., 2006).If similar conditions exist within fish, we expect energetically demanding behaviours, such as reproduction, to be reduced or cease prior to declines in absolute aerobic scope.Evidence of such trade-offs have been previously demonstrated in A. polyacanthus where reproductive output (i.e.clutch size × egg area) was reduced at temperatures >28.5 • C when fish were placed on a high-food diet and ceased on a low-food diet (Donelson et al., 2010).Additionally, when A. polyacanthus were acclimated to +3.0 • C for two generations, restoration of aerobic capacity was observed, but not reproduction (Donelson et al., 2016;Bernal et al., 2018) Collectively, results provide support for the multiple performance-multiple optima hypothesis (Clark et al., 2013) and highlights the need to study a range of performance fitness-related metrics.There is the potential that repeated PHA injections may allow for acquired immune response as previous research in blue-footed boobies (Sula nebouxii) detected an average increase of 90% between first and second PHA injections; attributing the increase to acquired T-mediated immunity (Santiago-Quesada et al., 2015).Thus, the increased swelling at 28.Evidence of co-gradient variation in aerobic capacity suggests that for the populations examined, phenotype and environmental influences are aligned.However, countergradient variation between A. polyacanthus populations was previously identified when comparing low-latitude (i.e.Lizard Island) and high-latitude (i.e.Heron Island) populations, which are further north and south than the populations examined in this study, respectively (Gardiner et al., 2010).Gardiner et al. (2010) sampled juvenile fish from shallow lagoons, whereas fish in this study were adults collected from ∼7 to 12 m on coral reef slope.Reef flats and lagoons generally experience greater thermal variability (minimums, maximums and magnitude of diurnal variation) via exposure to semidiurnal tidal oscillations compared to reef slopes that are exposed to the open ocean and hence more thermally stable, and this is true for the lagoon sites at Heron Island (Brown et al., 2023).Additionally, A. polyacanthus from Heron Island have been shown to have high capacity for phenotypic plasticity (Donelson and Munday, 2012;Ryu et al., 2018).In this study, evidence suggests that co-gradient thermal variation can be seen across populations; however, previous findings (Gardiner et al., 2010;Donelson and Munday, 2012) suggest a caveat that biogeographic differences (e.g.water depth, thermal variability) at local scales can disrupt larger biogeographical patterns.
Determining spatial patterns of thermal adaptation underpins the ability to predict population responses to climate change (Sorte et al., 2011;Moran et al., 2016).Climate envelope models frequently assign populations identical thermal tolerances; however, such approaches risk inaccurately projecting species trajectories by ignoring intrapopulation variation.Findings from this experiment demonstrated different oxygen consumption capacity amongst A. polyacanthus populations from low-and high-latitude regions as well as a decline in immune response and increased reliance on anaerobic pathways within both regions as temperatures warm.Evidence from previous research further suggests that predicting species responses to climate change would additionally benefit from considering the ability for populations to respond via plastic responses (Donelson et al., 2012), as well as differences in plastic potential between populations (Donelson and Munday, 2012).Furthermore, when results from this study are examined concurrently with Gardiner et al. (2010), it suggests that fine-scale biogeographic features can create pockets of adaptive heterogeneity, and that thermal tolerance may be tied closer to mean summer ranges rather than mean summer averages.These findings indicate that the adaptive landscape of species within marine environments may resemble a heterogenous mixture of populations with varying levels of adaptability.Therefore, it is necessary to sample populations from different environments to understand species' adaptive landscape.

Lay Summary
This study investigated the thermal performance of several traits from damselfish populations in two different regions on the Great Barrier Reef (low-and high-latitude).Oxygen consumption rates and aerobic enzyme activity at elevated temperatures were found to differ between the regions, while immunocompetence, haematocrit, and anaerobic enzyme activity were similar.

Figure 2 :
Figure 2: Thermal performance curves of A) resting oxygen performance, B) maximum oxygen performance and C) absolute aerobic scope of fish from low-(solid lines) and high-latitudinal (dashed line) regions across four different temperatures (i.e. 27, 28.5, 30 and 31.5 • C).Ribbon represents 95% confidence intervals.